Lipochito-oligosaccharides stimulating arbuscular mycorrhizal symbiosis

ABSTRACT

The invention relates to lipochitooligosaccharides obtainable from arbuscular mycorrhizal fungi, and which are useful for stimulating arbuscular mycorrhizal symbiosis, and lateral root formation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/126,942, filed Apr. 29, 2011, which is a continuation-in-part ofInternational Application No. PCT/IB2009/007492, filed in English onOct. 28, 2009, which designates the United States, and which claims thebenefit of PCT application No. PCT/IB2008/003484, filed in English onOct. 29, 2008, which also designates the United States. Each of theseapplications is incorporated by reference herein in its entirety.

The invention relates to lipochito-oligosaccharides involved inarbuscular mycorrhizal symbiosis, and to their applications.

Arbuscular mycorrhizal (AM) fungi have established symbioticassociations with plant roots for over 400 million years, since theappearance of the earliest land plants, suggesting that AM fungiassisted plants in their colonization of land (Remy et al., 1994). Thisgroup of fungi, recently renamed Glomeromycota, is one of the mostwidely distributed and AM associations are widely distributed throughoutthe plant kingdom including angiosperms, gymnosperms, pteridophytes andsome bryophytes (Smith and Read, 2008). Among the angiosperms, at least80% of the species can form AM symbioses, the only major exceptionsbeing Brassicaceae and Chenopodiaceae. AM fungi are able to transferrare or poorly soluble mineral nutrients such as phosphorus, zinc andcopper from the soil to the plant, which in turn provides carbohydratesto the fungus. This exchange of nutrients can be of critical importancewhen soil fertility and water availability are low, conditions thatseverely limit agricultural production in most parts of the world (Smithand Read, 2008).

Another known symbiotic association between plants and soilmicroorganisms is the rhizobial symbiosis. In contrast with thearbuscular mycorrhizal symbiosis, which is broadly distributed amongplants, the rhizobial symbiosis is restricted to legumes, and instead offungi, it involves nitrogen-fixing bacteria collectively calledrhizobia, which belong to several genera including Rhizobium,Bradyrhizobium, Azorhizobium, and Sinorhizobium. The rhizobial symbiosisresults in the formation of specific structures, the nodules, on theroots of the legume host. Nodules provide an appropriate environment forrhizobia, allowing them to fix molecular nitrogen and to providecombined nitrogen to the legume host. The initiation of theRhizobium-legume association depends on symbiotic signals that areproduced by both symbiotic partners. The signals released by the plantare usually flavonoids excreted in root exudates. These flavonoidsinteract with rhizobial transcription factors of the NodD family, whichactivate the transcription of nodulation (nod) genes involved in theproduction of the bacterial signaling molecules termed Nod factors(Dénarié et al., 1996). Nod factors share a common basic structureconsisting of a chitin backbone of four or five beta-1,4-linkedN-acetylglucosamine residues, N-acylated at the non-reducing end with afatty acid group of variable length and degree of unsaturation. Thisbasic structure can be further N-methylated at the nonreducing end, andcan also be O-substituted at the non-reducing end and/or at the reducingend. This variety of substituents provides a broad diversity of Nodfactors with different structures (for the description of diversestructures of Nod factors see Dénarié et al., 1996; D'Haeze et al.,2002). The specificity within the legume/rhizobial interaction (i.e. agiven species of rhizobia forms nodules on certain species of legumes)is the result of this diversity.

Genetic dissection of the pathway involved in Nod factor signaling inroots of the model legume Medicago truncatula has identified a number ofgenes involved in this pathway (Stacey et al., 2006). There is growingevidence that the Nod factor receptors are receptor-like kinases withextracellular sugar-binding LysM domains, such as those encoded by theNFP and LYK3 genes of M. truncatula. The interaction of a Nod factorwith its receptor induces a downstream signaling cascade, including therapid influx of calcium ions, calcium spiking, and expression ofspecific nodulin genes. These downstream events involve in particulargenes encoding proteins involved in calcium signaling, such as DMI1,DMI2 and DMI3 of M. truncatula which encode respectively a cationchannel, a leucine rich-repeat receptor-like kinase, and aCa²⁺/calmodulin-dependent protein kinase, and genes encoding proteinsinvolved in the control of gene expression, such as NSP1 and NSP2 whichencode transcription factors.

Although AM fungi are both agriculturally and ecologically extremelyimportant, the cellular and molecular mechanisms which control theformation of the mycorrhizal symbiosis, are far less known than thoseinvolved in rhizobial symbiosis.

It has been shown in M. truncatula that the nodulation and mycorrhizalprograms share at least three components (Catoira et al. 2000), namelythe products of the DMI1, DMI2 and DMI3 genes involved in calciumsignaling.

However, the events taking place upstream and downstream this calciumsignaling are still poorly characterized in the case of the arbuscularmycorrhizal symbiosis, in particular those involved in early signalingand leading to the recognition between the plant and the fungalpartners. The study of these events has been hampered by the facts thatthe fungal partner is an obligatory symbiont which cannot be grown inpure culture in the absence of living plants, and by the absence ofgenetic tools available for this group of fungi (Harrison, 2005).However it has been shown recently that diffusible signals are exchangedbetween the symbionts prior to physical interaction. On the plant side,compounds of the apocarotenoid family, strigolactones, can be secretedin root exudates and stimulate ramifications in hyphae from AM fungigerminating spores, signaling a physiological switch to activepre-symbiotic fungal growth (Akiyama et al., 2005; Besserer et al.,2006). On the fungal side, the existence of diffusible compoundsproduced by AM fungi and able to activate plant responses associated toendomycorrhization program, has also been reported (Kosuta et al., 2003;Weidmann et al., 2004; Navazio et al., 2007). More specifically, aseries of experiments performed with M. truncatula have recently shownthat AM fungi produce diffusible compounds that are able to stimulatethe expression of diverse plant responses. Three species of Gigasporaand one species of Glomus could trigger through a cellophane membranethe induction of expression of the MtENOD11 symbiotic gene in seedlingroots (Kosuta et al., 2003). Three fungal pathogens did not elicit thesame response, supporting the hypothesis that the response was inducedby a specific AM fungal signal molecule. Similarly, an AM fungus, Glomusintraradices, was shown to activate through a membrane the transcriptionof plant genes whose expression depends on the DMI3 symbiotic gene(Weidmann et al. 2004). In addition, a diffusible signal from AM fungiwas found to elicit a transient cytosolic calcium elevation in soybeancell cultures and the up-regulation of genes related to DMI1, DMI2 andDMI3 (Navazio et al., 2007).

Olah et al. (2005) reported that Nod factors from Sinorhizobiummeliloti, the rhizobial symbiont of M. truncatula, were able tostimulate mycorrhization and lateral root formation in M. truncatula.The stimulation of lateral root formation was also observed withdiffusible factors from arbuscular mycorrhizal fungi (Myc factors), butnot with Nod factors from rhizobial species (Sinorhizobium fredii andRhizobium leguminosarum), which cannot nodulate Medicago sp. They alsoreported that all the genes of the Nod factor signaling pathwaypresently identified, including in particular the NFP gene encoding theputative Nod factor receptor, as well as the DMI3 and NSP1 genes wererequired for stimulation of lateral root formation by Nod factors, butnot by Myc factors, which required only the DMI1 and DMI2 genes. On thebasis of these observations, these authors proposed a model explainingthe stimulation of mycorrhization and of lateral root formation inlegumes by both Myc factors and Nod factors. According to this model,Myc factors and Nod factors, which were recognized by different cellsurface receptors, activated a common DMI1/DMI2/DMI3 signalling pathway;in the case of Myc factors, DMI1 and DMI2 were sufficient forstimulation of lateral root formation, while DMI3 was required forstimulation of mycorrhization. Olah et al. also discussed the possiblechemical nature of the Myc factors. They hypothesized that they wereunlikely to be auxin-like compounds, since their effect on rootdevelopment was different from the one observed with these compounds.They also suggested that their structure should be different from thestructure of Nod factors, since they appeared to be discriminated by theNFP receptor.

Therefore, it appears that although the existence of diffusible “Mycfactors” produced by AM fungi, and able to activate plant responses, isrecognized in the art, the chemical nature of these factors has not beenidentified until now.

The inventors have now succeeded in purifying Myc factors from exudatesfrom both mycorrhized roots and germinating spores of the AM fungusGlomus intradices. They have further determined their chemicalstructure, and shown that they efficiently stimulate root systemdevelopment and root colonization by an AM fungus.

The Myc factors purified by the inventors are a mixture of sulfated andnon-sulfated lipochito-oligosaccharides (LCOs); they share with the Nodfactors a common basic chitin backbone of beta-1,4-linkedN-acetylglucosamine residues, N-acylated at the non-reducing end with afatty acid group. However the Myc factors have simpler structures thanthe Nod factors. The only O-substitution which is observed in Mycfactors is O-sulfation at the reducing end of the molecule. No otherO-substitutions such as O-carbamoyl at the non-reducing end, orO-fucosyl at the reducing end could be detected. The singleN-substitution on the non-reducing terminal GlcNAc residue for Mycfactors purified from Glomus intradices is the acylation by common fattyacids, mainly oleic (C18:1) and palmitic (C16:0) acids. In contrast theN-substitution of Nod factors is more complex. It is frequently a doublesubstitution by an N-methyl group and an N-acyl group (frequentlyvaccenic acid), as in rhizobial strains that nodulate most tropicallegumes and legumes of the Mimosoïdeae sub-family. N-methylation isspecified by the widespread nodS rhizobial gene (Dénarié et al., 1996).Alternatively, N-acylation by a specific poly-unsaturated fatty acid isthe rule among rhizobia that nodulate temperate legumes of the Galegoidclade (Dénarié et al., 1996). In fact, LCOs having a structure as simpleas the Myc factors characterized by the inventors were not observedamong the Nod factors synthesized by the various rhizobial strainsstudied so far (Dénarié et al., 1996; D'Haeze et al., 2002).

The invention provides a process for obtaining Myc factors from a fungusfrom the group Glomeromycota, wherein said process comprises obtainingexudates from plant roots mycorrhized with said fungus, or fromgerminating spores of said fungus, extracting said exudates withbutanol, and recovering the butanol extract containing saidlipochito-oligosaccharides.

According to a preferred embodiment of the invention, said processcomprises the further steps of subjecting said butanol extract to solidphase extraction on a C18 reverse-phase, with successive washes at 20%,50% and 100% acetonitrile and recovering the fraction eluted at 50%acetonitrile containing said Myc factors.

Still more preferably, said process comprises the further steps ofsubjecting said fraction eluted at 50% acetonitrile to reverse-phasehigh-performance liquid chromatography on a C18 reverse-phase column,using a linear gradient of 20% to 100% acetonitrile, and recovering thefraction eluted at 30-48% of acetonitrile which contains sulfatedlipochito-oligosaccharides, and/or the fraction eluted at 64-72% ofacetonitrile which contains non-sulfated lipochito-oligosaccharides.

According to a particular embodiment of the invention, said fungus fromthe group Glomeromycota is Glomus intraradices.

Fungal Myc factors can however also be extracted from other species ofGlomeromycota producing them, using the extraction steps disclosedabove, or variants thereof.

A “Myc factor” is herein defined a lipochito-oligosaccharide representedby the formula (I) below:

wherein n=0, 1, 2, 3, 4, or 5, preferably 2 or 3, R₁ represents a lipidsubstituent, containing 12 to 22, preferably 14 to 20 carbon atoms,which can be saturated, or mono-, di-, tri- tetra-, penta-, orhexaunsaturated, and R₂ represents H or SO₃H.

The lipid substituent R₁ is preferably a fatty acid chain. R₁ can alsorepresent an aromatic analogue of a fatty acid chain, as in Nod factoranalogues disclosed for instance by Grenouillat et al. (2004), or in PCTWO/2005/063784.

Advantageously, R₁ represents a chain of a fatty acid synthesized byarbuscular mycorrhizal fungi, in particular a C16 or C18 fatty acidchain, saturated, or mono- or di-unsaturated. Preferably, when saidfatty acid chain is unsaturated, it comprises at least onecis-unsaturation (for example the C18:1 oleic acid). By way ofnon-limitative examples of preferred fatty acid chains, one can mentionC16:0, C18:0, C16:1ω5, C16:1ω7, C18:1ω5, C18:1ω7, C18:1ω9, 18:2ω6,9,C20:0 iso, C20:1ω9 and C20:4ω6,9,12,15.

Myc factors can further be characterized and also differentiated fromlipochito-oligosaccharides of related structure such as Nod factors bytheir biological properties. These biological properties can be testedusing appropriate bioassays. In particular, one can use bioassays basedon the ability of the Myc factors to stimulate lateral root formation inthe model legume M. truncatula. More specifically, while Myc factorsshare with Nod factors the ability to stimulate lateral root formationin wild-type plants but not in the symbiosis-defective mutants dmi1,dmi2 and dmi3, Myc factors are also able, unlike Nod factors, tostimulate lateral root formation in the symbiosis-defective mutant nsp1.

If wished, bioassays for differentiating non-sulfated Myc factors fromsulfated Myc factors are also available (for instance if one wishes toseparate in a fungal extract, fractions containing non-sulfated Mycfactors from those containing sulfated Myc factors): for instance,sulfated Myc factors are able to induce the expression of the MtENOD11gene in growing roots of M. truncatula while non-sulfated Myc factorsare able to induce root hair branching in vetch.

Myc factors can be purified from fungi, as described above. They also beobtained by chemical synthesis and/or produced in genetically engineeredbacterial cells. For instance, a chito-oligosaccharide backbone,sulfated or not, can be synthesised in recombinant bacteria, asdisclosed for instance by Samain et al. (1997, 1999) for the synthesisof Nod factor precursors, and subsequently acylated on the free aminegroup of the non-reducing terminal sugar, as disclosed for instance byOhsten Rasmussen et al. (2004). One can also use a mutant strain of aRhizobiaceae bacterium producing Myc factors rather than Nod factors,for instance a strain genetically modified in order to express, amongthe structural genes of the Nod biosynthetic pathway, only thoseinvolved in the synthesis of the chito-oligosaccharide backbone andthose involved in the N-acylation of the non-reducing terminalglucosamine by an appropriate C16 or C18 fatty acid, and optionallythose involved in the O-sulfation of the reducing terminal glucosamine,as disclosed for instance by Ardourel et al. (1994), or Lugtenberg etal. (1995).

The invention also encompasses mixtures of different Myc factors offormula (I), and in particular mixtures of sulfated and non-sulfated Mycfactors, comprising one or more lipochito-oligosaccharides of formula(I) wherein R₂ represents H, and one or more lipochito-oligosaccharidesof formula (I) wherein R₂ represents SO₃H. Thelipochito-oligosaccharides of said mixture may further differ betweenthem by the number of N-acetylglucosamine residues and/or the nature ofthe substituent R₁ (for instance the length and/or the degree ofunsaturation of the fatty acid chain).

Mixtures of Myc factors of the invention can for instance be obtained byextracting Myc factors from arbuscular mycorrhizal fungi, as describedabove, and recovering the fungal extract. They can also be obtained byproducing separately the different Myc factors and mixing them.

Purified or synthetic lipochito-oligosaccharides and more specificallythe purified or synthetic Myc factors of formula (I) or mixtures thereofdescribed herein can be used to stimulate mycorrhization, and thus havea broad range of applications in agriculture, horticulture and forestry,for most cultivated plants which can establish mycorrhization, andtherefore possess Myc factor receptors.

In addition to their use for stimulating the arbuscular mycorrhizalsymbiosis, the purified or synthetic Myc factors or mixtures thereof canalso be used:

-   -   to stimulate the germination of seeds, which can be useful for        seed treatment with a broad range of applications in        agriculture, horticulture and forestry;    -   to stimulate the root system development, which is beneficial to        improve water and mineral nutrition.

They can be used for instance for treating seeds, or added to inoculantscontaining arbuscular mycorrhizal fungi, or added to the soil or theculture substrate of the plant. The purified or synthetic Myc factors ofthe invention can be used with any plant namely with plants which canestablish mycorrhization, including as well legumes as non-legumeplants, and including as well dicotyledons as monocotyledons, such ascereals. They can be used for plants grown under growth chamber, as wellas in greenhouse or in field conditions.

They can also be used for stimulating mycorrhizal colonization in theproduction of mycorrhizal inoculants (i.e. AM fungal spores or hyphae,or fragments of mycorrhized roots), as additive to the culture mediawhich are used for the production of these inoculants by plants grown onsoil or on hydroponic or aeroponic conditions, or by co-culture ofmycorrhizal fungi with excised roots.

The invention also encompasses compositions containing purified orsynthetic Myc factors or mixtures thereof, and an agriculturallysuitable carrier. Compositions of the invention may also comprise mutantstrains of Rhizobiaceae bacteria genetically modified in order toproduce Myc factors rather than Nod factors, as described above.Preferred compositions are those containing a mixture of sulfated andnon-sulfated Myc factors.

The Myc factors can optionally be combined with other activeconstituents, such as flavonoids, apocarotenoids such as strigolactones,or jasmonate which are plant compounds which have been reported to actas symbiotic signals (Harrison, 2005; Akiyama et al., 2005; Besserer etal., 2006).

The formulation of these compositions depends on the intended mode ofapplication, (for instance coating seeds, adding to a culture medium forproduction of mycorrhizal inoculants, treating the plant of the soil).They can for instance be formulated as water-dispersible orwater-soluble solids such as powders, granules, pellets, or films, asliquid aqueous solutions, suspensions, or emulsions, or as gels.

According to a preferred embodiment, these compositions are associatedwith fungal and/or plant material, for instance with an inoculant of anarbuscular mycorrhizal fungus, or with seeds of a plant able toestablish mycorrhization; advantageously, said seeds are coated with thecomposition.

Advantageously, the Myc factors are used in the composition at aconcentration of 10⁻⁵ M to 10⁻¹² M. When added to a culture medium forproduction of AM fungal spores, they can be used at a concentration of10⁻⁶ M to 10⁻¹⁰ M, preferably at a concentration of 10⁻⁷ to 10⁻⁹ M inthe medium. When used for seed treatment or for stimulating the rootsystem development, they can be used at a concentration of 10⁻⁶ M to10⁻¹⁰ M, preferably at a concentration of 10⁻⁷ to 10⁻⁹ M. When a mixtureof sulfated and non-sulfated Myc factors is used, concentrations as lowas 10⁻⁸ to 10⁻¹⁰ M can be used.

The invention will be understood more clearly with the aid of theadditional description which refers to the examples below and to theappended drawings. It should be clearly understood, however, that theseexamples and drawings, are given solely as illustration of the subjectof the invention and do not constitute in any manner a limitationthereof.

LEGENDS OF THE DRAWINGS

FIG. 1. Biological assays used to detect AM fungal symbiotic signals

a. The MtENOD11 assay. Roots of transgenic M. truncatula Jemalong A17seedlings carrying the reporter construct pMtENOD11-GUS. GUS activity isdetected by histochemical staining with5-bromo-4-chloro-3-indolyl-b-glucuronic. (1) Control roots treated withacetonitrile 2.5%. (2) Fraction after SPE and elution with 50%acetonitrile diluted 40 times. (3) The same fraction with a furtherten-fold dilution.

b1-b2. The VsHab assay. Root hairs of vetch (Vicia sativa subsp. nigra)observed under light microscope after staining with methylene blue. (b1)Root hairs treated with an inactive fraction are straight. (b2) Roothairs treated with active fractions are clearly branched.

FIG. 2. Semi-preparative C18 reverse phase HPLC profile of extracts frommycorrhized root exudates.

The initial isocratic phase with 20% acetonitrile lasted 10 min and wasfollowed by a 20-100% acetonitrile gradient for 20 min. The profilereveals the abundance of contaminating material present in mycorrhizedroot exudates. Fractions were collected every two minutes and weretested for biological activity on MtENOD11 and VsHab. Horizontal barsindicate the retention time of compounds in fraction A that are activeon MtENOD11, and of compounds in fraction B, more hydrophobic, that areactive on VsHab.

FIG. 3. Semi-preparative C18 reverse phase HPLC profile of extracts fromgerminating spore exudates.

The chromatographic conditions are the same as in FIG. 2. The profilereveals that spore exudates contain much less contaminating materialthan mycorrhized root exudates. Fractions were collected every twominutes and were tested for biological activity on MtENOD11 and VsHab.Horizontal bars indicate the retention time of compounds in fraction Athat are active on MtENOD11, and of compounds in fraction B, morehydrophobic, that are active on VsHab.

FIG. 4. Influence of mild methanolic hydrolysis on the biologicalactivity of fraction A.

Mild methanolic hydrolysis has been reported to remove the sulfatemoiety of sulfated LCOs without altering other structural features ofthese molecules. Fraction A collected during semi-preparative HPLC ofgerminating spore exudates was mildly hydrolyzed and tested forbiological activity on MtENOD11 and VsHab assays. Biological activity isrepresented by vertical bars. Whereas unhydrolyzed fraction A is activeon MtENOD11 and inactive on VsHab, the hydrolyzed fraction has lostactivity on MtENOD11 and gained activity on VsHab. These data indicatethat the biological activity of fraction A on the MtENOD11 assay is dueto the presence of sulfated LCOs.

FIG. 5. Tetrameric sulfated LCOs N-acylated by C16 fatty acids.

UPLC/MS traces, in the negative mode, of the fraction 4 isolated aftersemi-preparative C18 HPLC. Extracted ion currents corresponding tosulfated tetramers and corresponding spectra are given. This figureindicates that compounds responding at m/z 1101.5, 1103.5 and 1105.5 areeffectively present in the samples. These m/z correspond to sulfatedtetrameric LCOs N-acylated by C16:2, C16:1 and C16:0 respectively.Regarding the relative intensities of the three, it appears that 1105.5(LCO-IV-C16:0) is the most abundant, followed by 1103.5 (LCO-IV-C16:1).

FIG. 6. Tetrameric sulfated LCOs N-acylated by C18:1 fatty acid.

UPLC/MS traces, in the negative mode of the fraction 5 isolated aftersemi-preparative C18 HPLC showing that the most abundant compound (m/z1135.5) is N-acylated by a C18:1 fatty acid.

This profile also indicates that no LCO bearing a C18:0 fatty acid ispresent in this fraction (m/z 1133.5) as this ion is only the isotope +2of the LCO bearing the C18:1 chain. As the second mass spectrumdemonstrates, the di-unsaturated C18-LCO is a very minor compound.

FIG. 7. Pentameric sulfated LCOs N-acylated by C18:1 fatty acyl.

This profile shows that lipochitopentamers are also present, butcompared to the corresponding tetramers (see FIG. 5) they areapproximately 30 times less abundant. The LCO-V-C18:1 can be detected.

FIG. 8. Checking for the presence or absence of a given compound.

When the requested mass does not correspond to ions present in thesample the profile instead of giving a single peak gives a very largenumber of background peaks. The very complex profile obtained with ioncurrent m/z 1332.6 demonstrates the absence of a C18:2 chitopentamer inthe sample. In contrast, the clear single peak observed with ion currentm/z 1334.6 clearly shows the presence of a C18:1 pentamer.

FIG. 9. Comparison of fragmentation pattern by MS/MS of major sulfatedMyc factor and S. meliloti Nod factor

Demonstrating the presence of compounds having the adequate mass at theexpected HPLC retention time, is not sufficient to attest theirstructure. Therefore, we performed MS/MS analysis of the major sulfatedMyc compound. This figure presents the comparison between the S.meliloti sulfated tetrameric Nod factor N-acylated by C 16:2 in thenegative mode MS/MS and the one recorded on the major tetrameric “Mycfactor” present in the sample. Characteristic ions of the reducing endat m/z 503 (Y₂), 605 and 706 (Y₃) are clearly detected in both case aswell as the characteristic neutral loss of 101 amu (intracyclic rupture)starting from the molecular ion. The perfect fit between the twofragmentation patterns indicates the structural affiliation of the twomolecules.

FIG. 10. Effect of Myc extract fractions on lateral root formation in M.truncatula

(A) The AM fungal signal that stimulates LRF is amphiphilic.

Comparison of the effect of aqueous (Aq), butanol (BuOH) and ethylacetate (EA) extracts of germinating spore exudates (GSP24) on M.truncatula A17. The butanol extract stimulates LRF from day 5 on(significant at P<0.05), whereas the aqueous and acetyl acetate extractsare not active.

(B) LRF stimulation is mediated via the DMI symbiotic signaling pathway.

Comparison of the effect of mycorrhized root exudate (MRE1) butanolextracts, further purified by SPE eluted with 50% acetonitrile, on M.truncatula wild-type (A17) and on a dmi1 mutant (Y6). The Myc extractstimulates LRF on the wild-type but not on the dmi1 mutant.

(C) Both fractions A and B stimulate LRF.

Fractions A et B were collected after semi-preparative HPLC ofmycorrhized root exudates (MRE-1). Fraction MRE-1 A contained sulfatedLCOs and fraction MRE-1 B contained non-sulfated LCOs. Both fractionsstimulated LRF significantly (P<0.05).

FIG. 11. Effect of a mixture of sulfated and non-sulfated Myc factors onmycorrhization of Medicago truncatula.

a. Mycorrhization in axenic conditions. Plants were grown in test tubeson gellified slopes of M medium in which Myc factors were incorporatedat a 10⁻⁸ M concentration. 50 sterile spores (Glomus intraradices) werelaid close to seedling roots. Extent of mycorrhization was measured bycounting the number of infection units six weeks after inoculation.Results were analyzed by the non-parametric Kruskal-Wallis statisticaltest.

b. Mycorrhization in non-sterile conditions. Plants were grown on asubstrate made of charred clay granules, inoculated with 50 sterilespores of G. intraradices, Myc factors being added to the nutrientsolution at a concentration of 10⁻⁸ M. Three weeks after inoculationroot colonization was estimated by the grid intersect method.

FIG. 12. Effect of Myc factors on root architecture in Medicagotruncatula.

a. Effect on lateral root formation. Histogram showing the effect of amixture of both sulfated and non sulfated Myc factors (NS+S), sulfatedMyc Factors (S) and non sulfated Myc factors (NS) at 10⁻⁸ M, 10⁻⁹ M and10⁻¹⁰ M concentrations on the lateral root formation of M. truncatulawild-type (A17), eight days after treatment.

Forty plants were used per experiment and statistical analysis was madeby the Student's t-test between control and treated plants.

b. Effect on total root length. Histogram showing the effect of amixture of sulfated and non-sulfated Myc factors on the total rootlength of seedlings. Seedling were grown for eight days, roots were cutand the root system was scanned and measured by the WinRhizo software.Data were analyzed by the Kruskal-Wallis test.

(*) and (**) denote respectively a significant (P<0.05) or a highlysignificant (P<0.01) difference and bars represent the standard error ofthe mean (SEM).

FIG. 13. Genetic analysis of the Myc factor-activated signalling pathwayleading to stimulation of lateral root formation.

Histogram showing the effect of the non-sulfated Myc factor (10⁻⁸ M) onlateral root formation of M. truncatula wild-type (A17) and symbioticsignalling pathway dmi1, dmi2, dmi3 and nsp1 mutants. Means arerepresented as percentage of the control value eight days aftertreatment.

For each genotype, data from at least two independent experiments with40 plants per experiment were pooled and statistical comparisons weremade using the Student's t-test between control and each treatment. (**)indicates a highly significant (P<0.01) difference and bars representthe standard error of the mean (SEM).

FIG. 14. Effect of Myc factors on in vitro mycorrhizal colonization ofexcised transformed roots of carrot.

a. Effect of a mixture of bacterial sulfated and non-sulfated Mycfactors. Roots were inoculated with sterile spores of G. intraradices(10 spores/mL of growth medium) and treated once a week during threeweeks with or without a mixture of Myc factors at 10⁻⁸M. The mycorrhizalcolonization rate was observed after six weeks. (**) denotes a highlysignificant difference with control (Student's t test, P-value<0.01).Vertical bars represent the standard error of the mean (SEM).

b. Effect of a mixture of synthetic sulfated and non-sulfated Mycfactors. Roots were inoculated with sterile spores of G. intraradices(100 spores/mL of growth medium) and treated once a week during fourweeks with or without a mixture of Myc factors at 10⁻⁸M. The mycorhizalcolonization rate was observed after eight weeks. (*) denotes asignificant difference with control (Student's t test, P-value=0.0119).

FIG. 15. Effect of Myc factors on mycorrhization of Tagetes patula.

a: Effect of a mixture of sulfated and non-sulfated Myc Factors on thenumber of infection units per plant (a1), root length (a2) and densityof infection (a3). Plants were inoculated with about 100 sterile sporesof Glomus intraradices and treated twice a week during three weeks withor without Myc factors at 10⁻⁸M. The number of infection units, rootlength and the density of infection units were determined after fourweeks. (**) denotes a highly significant difference with control(Student's t test, P-value=0.004086).

b: Effect of sulfated (S), non-sulfated (NS) or a mixture of bothsulfated and non-sulfated (NS+S) Myc Factors on the mycorrhizal rootcolonization. Plants were inoculated with about 100 sterile spores of G.intraradices and treated twice a week during three weeks with or withoutMyc factors at 10⁻⁸M. The colonization rate was measured after fourweeks.

FIG. 16. Effect of Myc factors on germination of tomato seeds.

Effect of non-sulfated (NS), sulfated (S), and a mixture of bothsulfated and non-sulfated (NS+S) Myc Factors on germination of tomatoseeds at 14° C.

a. Myc factors were added to germination plates at 10⁻⁸M, 10⁻⁹M and10⁻¹⁰M. Germination rate was scored everyday. Results were analyzed withthe Kruskal-Wallis test. (***) and (**) denotes respectively a veryhighly (P-value<0.001) and highly (<0.01) significant difference withcontrol, and vertical bars represent the standard error of the mean(SEM).

Effect of a mixture of sulfated and non-sulfated Myc factors on seedgermination at 14° C.

b1. Kinetics of germination. Myc factors were added at 10⁻¹⁰M. Resultswere analyzed with the non-parametric Kruskal-Wallis test. After day 6,differences were highly significant. Vertical bars represent thestandard error of the mean (SEM).

b2. Photograph of representative germination plates with and without Mycfactors ten days after sowing.

MATERIALS AND METHODS

Natural Sources of Myc Factors

The AM fungus Glomus intraradices strain DAOM 197198, which has beenmaintained in co-culture with excised roots for many years (Chabot etal., 1992), is well characterized and its genome is being sequenced.This strain is used by PREMIER TECH company for the industrialpreparation of commercial inoculants and for the production of purifiedspores for research purpose. For example, these purified spores wereused as a source of DNA for the project of G. intraradices genomesequencing. We used two sorts of exudates, both prepared from materialspurchased from PREMIER TECH BIOTECHNOLOGIES (Rivère-du-Loup, Québec,Canada):

(i) Exudates from mycorrhized roots (EMR). Mycorrhiza production wasachieved by co-cultivation of G. intraradices with excised transformedroots of carrot. The growth medium was solidified with Phytagel. Afterappropriate growth of the mycorrhized roots, the gel was liquefied byadding sodium citrate as chelating agent, and the liquid EMR wasconditioned in 4 liter containers which were stored at 4° C.

(ii) Exudates from germinating spores (GSP). Purified sterile spores ofthe AM fungus Glomus intraradices were conditioned by bottles containingapproximately one million of spores. Bottles were stored at 4° C. Sporeswere germinated at 30° C. in a 2% CO₂ incubator for 10 days.

Bioassays Used for Purification of Myc Factors

To detect the presence of AM fungal symbiotic signals during the varioussteps of extraction and purification, three bioassays were used. (i) TheM. truncatula ENOD11::GUS construct was shown to be induced duringmycorrhiza formation and by a diffusible compound from diverse AM fungi(Journet et al., 2001; Kosuta et al. 2003) (=MtENOD11 assay). (ii) Thelateral root formation in M. truncatula was shown to be stimulated by adiffusible compound from diverse AM fungi and the response required theDMI symbiotic signaling pathway (Olah et al., 2005) (=MtLRF assay).(iii) In addition we used a modified Vicia sativa (vetch) root hairbranching assay which allows the detection of various non-sulfated LCOs(=VsHab assay).

(i) Induction of the Symbiotic MtENOD11 Gene in Transgenic Medicagotruncatula.

We have previously shown by experiments in which the AM fungus wasseparated from the plant root by a cellophane membrane that a diffusibleAM fungal compound can induce the expression of an MtENOD11promoter-gusA transgene in growing lateral roots of M. truncatula(Kosuta et al., 2003). The protocol used was as previously described(Andriakaja et al., 2007) with the following modifications: no paperdisc was inserted on the top of the agar plate and the treatment wasmade by addition of 40 microliters per seedling. To check whether theENOD11 response was induced via the DMI signaling pathway, we comparedthe response observed in the M. truncatula wild-type line A17 and in amutant line carrying a mutation in the DMI1 gene (Y6 mutation).

(ii) Root Hair Branching of Vetch

Vetch (Vicia sativa subsp. nigra) is a small seeded legume which isconvenient for the microscopical observation of root hair deformations.Vetch root hair deformations are elicited not only by the Nod factors ofthe specific bacterial symbiont Rhizobium leguminosarum bv. viciae butalso by a variety of non-sulfated Nod factors (Roche et al., 1991; Priceet al., 1992).

This assay is thus appropriate to detect the presence of non-sulfatedLCOs. In previous reports the assay was done in a liquid medium. We havedevised an assay on agar plate which is more sensitive and reproducible.Seeds were first sterilized in sulfuric acid for 20 min, rinsed twicewith sterile water, and then treated for 20 min in calcium hypochlorite(5 g/150 ml after paper filtration) and rinsed five times with sterilewater. Seeds were left in water overnight at 4° C., transferred ontosoft agar plates and incubated three days at 4° C. to increase thehomogeneity of germination. Plates were then left for 36 hours at 22° C.in the dark for germination. Five young seedlings (root length ofapproximately 1 cm) were sown in Petri dish, on Fahraeus agar plates,surrounded with parafilm, and left three days, in a vertical position ina growth chamber at 22° C. When roots became hairy, 40 microliters ofthe solution to be tested were gently deposited along the roots, andseedlings were grown for 30 hours at 22° C. For root hair branchingobservation, roots were sectioned, inserted between a slide and acover-slip in a 0.02% methylene blue solution, and observed under alight microscope. Ten plants were observed per treatment.

Mycorrhization Assays

Sources of AM fungal inoculum for mycorrhization experiments weresterile spores of Glomus intraradices, either purchased from PremierTech Biotechnologies Ltée (Rivière-du-loup, Québec, Canada) or producedon excised transformed carrot roots as described by Bécard and Fortin(1988). Mycorrhized transformed carrot roots were grown as described inChabot et al. (1992) and subcultured every ten weeks on M medium (Bécardand Fortin, 1988) gellified with 0.4% Phytagel (Sigma). Aftersolubilization of Phytagel with citrate buffer (Doner and Bécard, 1991),spores were collected as described under sterile conditions and storedat 4° C. in Ultrapure water for at least four weeks before use.

Mycorrhization tests were carried out on three plant species, the modellegume M. truncatula and two non-legumes, carrot (Daucus carota,Umbelliferae family) and French marigold (Tagetes patula, Asteraceaefamily).

Myc factors were dissolved in water/acetonitrile (50/50) to prepare a10⁻³ M stock solution, which was then diluted to the appropriateconcentration with water or growth medium. The same amount ofacetonitrile solvent traces was added to control plates.

In vitro Mycorrhization of Excised Transformed Carrot Roots

Sterile excised transformed carrot roots were grown on M mediumsolidified by 0.4% phytagel, at 24° C. in the dark, and subculturedevery ten weeks (Chabot et al., 1992). Roots were collected bysolubilization of Phytagel with citrate buffer (Doner and Bécard, 1991)and washed with sterile deionised water. Plates for mycorhization assaywere prepared as follows: in Petri dishes (Ø 90 mm) a first layer of 20ml M medium containing 0.3% Phytagel was poured and left forsolidifying. A second layer of the same medium was then pouredcontaining 20 or 200 spores/ml and Myc factors at the appropriateconcentration. In control plates Myc factor solution was replaced by thesame volume of the medium used for preparing the Myc factor solution.Root fragments were laid on the medium surface with approximately thesame amount (number of fragments and root length) in the differentplates. Dishes were closed with Parafilm tape and incubated in the dark,in a growth room at 24° C. and 50% humidity, during six or eight weeks.Myc factors were added once a week on the plate surface during the threeor four first weeks for experiments of six or eight weeks respectively.To observe fungal colonization, roots were collected after liquefactionof phytagel by citrate buffer, washed and stained by the ink-vinegarmethod (Vierheilig et al., 1988). Colonization rate was estimated by thegrid intersect method (Giovanetti and Mosse, 1980).

In vivo Mycorrhization of Tagetes patula

Seeds of Tagetes patula, var. Légion d'honneur, were obtained fromCaillard (84091 Avignon, France). Seedlings were grown four weeks in 50ml Falcon tubes filled with a substrate made of washed and autoclavedclay (charred granular Montmorillonite; ref “Oil Dry US Special”,Brenntag Bretagne, ZI de Tory, BP41, Avenue des Ferrancins, 71210Montchanin). To ensure watering of the seedlings, tubes were piercedwith three small holes at the bottom, and individually placed in 120 mlplastic boxes (5.5 cm diameter/7 cm high), closed with an opaque cappierced to receive and fix the Falcon tubes.

Boxes were filled with 80 ml water and wrapped with aluminium foil. TheFalcon tubes clay substrate was hydrated with 20 ml Long Ashton lowphosphate solution (Hewitt et al, 1966). In each tube one seed wasplaced underneath the surface of the substrate, and a hundred of fungalspores were dropped around the seed, in 1 ml 10⁻⁷ M Myc factor orcontrol solution. Each plant received 1 ml 10⁻⁷ M Myc factor, or 1 mlcontrol solution, twice a week for three weeks. Pots were placed in agrowth chamber, at 25° C., with a 16 h photoperiod and a light intensityof 180 μEinstein·m⁻²·s⁻¹.

Two series of experiments were done. In the first, a mixture of sulfatedand non-sulfated synthetic Myc Factors was tested with 12 seedlings pertreatment. In the second, sulfated, non-sulfated and a mixture of bothwere tested with 20 seedlings per treatment. Plants were harvested after4 weeks. The inner root system was stained with Schaeffer black ink(Vierheilig et al, 1998). Quantification of root colonization by thefungus was performed under a binocular magnifying glass, and two methodswere used: (i) for the first experiment, the number of infection units(zones containing arbuscules, vesicules and internal hyphal networks)was counted for each plant, and (ii) for the second, the percentage ofroot length colonized by the fungus, that is, showing arbuscules,vesicules or both, was determined by the gridline intersect method(Giovannetti et al, 1980).

Mycorrhization of Medicago truncatula in Axenic Conditions

Plants were grown in test tubes on slopes of 20 ml gellified MM medium(Olàh et al, 2005) as described in Ben Amor et al. (2003). Myc factorsat 10⁻⁸ M concentration (or control solution) were incorporated directlyinto the sterile medium. Fifty sterile spores of G. intraradices wereput at the bottom of each slope near the seedling root. Test tubes wereplaced in a growth chamber at 25° C. with a 16 h photoperiod and lightintensity of 366 μEinstein·m⁻²·s⁻¹. After six weeks, the root systemarchitecture was analysed by the Winrhizo Scientific Software(Instruments Regent Inc, 2672 Chemin Ste Foy RD, Sainte Foy, Quebec,Canada). Quantification of root colonization was done by direct countingof infection units under a binocular glass magnifier, after rootstaining by the ink-vinegar method (Vierheilig et al, 1998).

Mycorrhization of Medicago truncatula on a Charred Clay Substrate

Germinated seedlings were grown for three weeks in 50 ml Falcon tubes asdescribed above for Tagetes mycorrhization. Twenty G. intraradicesspores were dropped around the seedling roots, in 1 ml of 10 ⁻⁷ M Mycfactor or control solution. Then each plant received 1 ml of 10⁻⁷ M Mycfactor, or 1 ml control solution, twice a week during two weeks. Twoseries of experiments were done with 12 seedlings per treatment. Potswere placed in a growth chamber, at 25° C., with a 16 h photoperiod, andlight intensity of 366 μEinstein·m⁻²·s⁻¹.

Plants were harvested after 3 weeks. The inner root system was stainedwith Schaeffer black ink (Vierheilig et al, 1998). The percentage ofroot length colonized by the fungus, that is, showing arbuscules,vesicules or both, was determined by the gridline intersect method(Giovannetti et al, 1980)

Bioassays Used for Testing the Developmental Activity of Myc Factors

Bioassays were devised to study the developmental activity of purifiedor synthetic Myc factors.

(i) Stimulation of Root System Development in the Model Legume M.truncatula.

We have previously shown that a diffusible factor from AM fungistimulates the lateral root formation (LRF) in M. truncatula via the DMIpathway (Olah et al., 2005). We have used this bioassay to test thedevelopmental activity of purified Myc factors. The protocol used was asdescribed previously except that vitamins were not added to the Mmedium.

Identification of plant genes involved in Myc factor signaling wasperformed in M. truncatula, using the genetic analysis of the LRFresponse already described (Olah et al., 2005). LRF responses to Mycfactors were studied in the wild-type M. truncatula Jemalong A17 line,as a control, and in the symbiosis-defective mutants dmi1 (Y6), dmi2(TR25), dmi3 (TRV25), and nsp1 (B85).

(ii) Tomato Seed Germination

Seeds of tomato variety Heinz 1706 were from the Core collection oftomato seeds of INRA. They were kindly provided by Rend Damidaux of the“Génétique et Amélioration des Fruits et Légumes” laboratory at INRA84143 Montfavet cedex (France). From this core collection sample, seedswere multiplied at LIPM (INRA-CNRS, Toulouse). Seeds were stored at 4°C. Seeds were sterilized for 45 min in a filtered solution of 0.262 Mcalcium hypochlorite (2.5 g of CaOCl2 in 75 ml water), to which twodrops of Tween 20 had been added. Hypochlorite solution was removed andseeds were rinsed three times with sterile distilled water. Germinationagar plates were prepared by dissolving 9.375 g of Difco Agar Granulated(Becton-Dickinson) in one liter of distilled water. A solution of 10⁻³ MMyc factor was prepared in 50/50 water/acetonitrile, and was thendiluted to the appropriate dilutions with water. The same amount ofacetonitrile solvent traces was added to the control plates. Fifteenseeds were laid by plate, with six or eight repeats per treatment.Plates were incubated in the dark at 14° C., 20° C. and 28° C.Germination rate was scored everyday.

Statistical Analysis of Data.

Data of biological assays were statistically analyzed with the Student'st-test or analysis of variance for data following a normal distributionand having homogeneous variances, and Kruskal-Wallis or Wilcoxonnon-parametric tests for non-normal distributions. Statistical softwarewas from the R system (R Development Core Team, 2009).

Biochemical Analyses

Liquid/Liquid Extraction for Mycorrhized Roots Exudates:

In a two liter bulb, 1.6 liter of mycorrhized root exudates wasextracted a first time with 400 ml (¼ of volume) butanol (1-butanol or2-methyl-1-propanol) and the mixture was left for decantation to get aclear butanol phase with a thin interphase, permitting a good separationof the aqueous and butanol phases (at least six hours). The aqueousphase was then extracted a second time with 350 ml (approximately ⅕ ofvolume) butanol and left overnight. After this second extraction, thetotal butanol phase (extraction 1 and extraction 2) was evaporated to avolume of approximately 0.5 liter, which was washed by a liquid/liquidextraction with the same volume of bi-distilled water. The washedbutanol phase was evaporated, transferred in a small balloon and driedusing a rotary evaporator. The dry extract was then re-dissolved in 5 mlwater/acetonitrile (1/1) and filtered on cotton (preliminary washed withchloroform) in a 8 ml glass tube and then dried under nitrogen flux.

Liquid/Liquid Extraction for Germinating Spores Exudates:

Exudates from one million of germinating spores (approximately 150 ml)were first extracted with ⅓ of volume of ethyl acetate. The mixture wasleft for decantation to get a thin interphase and a good separation ofthe aqueous and ethyl acetate phases (at least six hours). The aqueousphase was extracted a second time with ⅓ of volume of ethyl acetateovernight. The aqueous phase was then extracted with butanol (1-butanolor 2-methyl-1-propanol) following the same steps as for the ethylacetate extraction. The butanol and ethyl acetate phases volumes werereduced to few ml using a rotary evaporator. Each phase was transferredinto a 5 ml tube and dried under nitrogen flux.

Purification by Solid Phase Extraction (SPE):

Column preparation: The SPE system was made up of a Chromabond 3 mlglass column filled with C18 reverse phase (SUPELCO Discovery DSC-18). Afirst glass fiber filter was introduced at the bottom of the column. Thesolid phase was added into the column to represent 3.5 cm height in thecolumn. A second glass fiber filter was laid on top of the solid phaseand pushed in to compress the solid phase. Before use, the column waswashed with acetonitrile (ACN) and with water, and then conditioned withacetonitrile (ACN) 20% in water.

Pre-filtration: Extract was dissolved in one ml of 20% ACN. The extractwas filtered on cotton in a Pasteur pipette (preliminary washed withchloroform) and deposited on the C18 column. The tube and the filterswere rinsed with 1.5 ml of ACN 20%.

Chromatography: Using a syringe, the extract was pushed through the C18phase. The running out liquid of the column was collected in an 8 mlglass tube. To get rid of the non-adsorbed compounds, the phase wasabundantly washed (equivalent 5 times the volume of the solid phase with20% ACN in water). This volume was recovered in the same tube. Thenmolecules retained on the column were eluted with a 50% solution of ACNin water. The elution volume being equivalent to 5 times the solid phasevolume was recovered in a second glass tube. Finally the stronglyadsorbed molecules were eluted from the column with 100% ACN. The volumeof solvent (about 6 ml) was recovered in a third tube. The threesolutions (20%, 50% and 100% of ACN) were evaporated under nitrogenflux, in order to get dry residues. Each residue could then bere-dissolved in the volume appropriate to realize the semi-preparativeHPLC. A SPE column was used to purify approximately 5 liters ofmycorrhized root exudates.

Semi-Preparative HPLC:

Purification was performed on a High-Performance Liquid ChromatographyShirnadzu LC10 separation module (Shimadzu corporation, Kyoto, Japan)with a semi-preparative C18 reverse phase column (8 mm×250 mm; 5 μm,Equisorb, CIL-Cluzeau). The injection loop had 100 microliter volume.The chromatographic procedure was the following: for 10 min in isocraticmode with solvent A (20% acetonitrile in water), followed by a lineargradient for 20 min from solvent A to solvent B (100% acetonitrile) andanother isocratic step at 100% acetonitrile for 5 min. Two minutes arenecessary to come back to the initial conditions (20% ACN). The flowrate was 2 ml min⁻¹ and UV absorption was monitored at 206 nm.Collection of samples along the gradient was done every minute (2 ml)resulting in 14 fractions.

Supplementary Analytical HPLC for Detection of Non-Sulfated LCOs

Purification was performed on a High-Performance Liquid ChromatographyShimadzu LC10 separation module (Shimadzu corporation, Kyoto, Japan)with a C8 phase column (Zorbax XDB-C8 HP-Eclipse (Hewlett Packard) 5 μm,4.6×150 mm) for 5 min in isocratic mode with 30% methanol in watersolvent, followed by a linear gradient for 20 min to the 100% methanolsolvent, followed by another isocratic step at 100% methanol for 5 min.2 min were necessary to come back to the initial conditions. The flowrate was of 1 ml min⁻¹ and the UV absorption was monitored at 206 nm.Collection of samples occurred along the gradient and isocratic step at100% methanol every minute (about 1 ml) from 15 to 23 min producing 8fractions.

UPLC-ToF MS Analyses:

Each fraction collected from semi-preparative HPLC was submitted toUPLC-MS analysis on an Acquity UPLC coupled to Q-Tof Premier massspectrometer (Waters Corporation). The UPLC column was an Acquity column(2.1 mm×10 cm, 1.7 μm) (Waters, USA), and the flow rate was 0.45 ml/min.For the more hydrophilic compounds (semi-preparative HPLC fractions 1 to9) the program was a linear gradient ranging from 10% ACN (in 1% aceticacid/water) to 100% ACN within 7 minutes, followed by an isocratic stepat 100% ACN for 2 min and then a return to the initial conditions (2min) and finally a reconditioning step of 1 min with 10% ACN (in 1%acetic acid/water). To get a better resolution of the more hydrophobiccompounds (semi-preparative HPLC fractions 6 to 11) the UPLC gradientwas more extended: linear gradient starting at 25% ACN in 0.1% aceticacid/water and reaching 100% ACN within 7 minutes. For the massspectrometer, capillary was set to 3.2 kV and the cone to 10 V. Internallock mass was performed by continuous introduction into the source of aLeucine-enkephalin solution. Spectrometer was calibrated before eachexperiment. The more hydrophilic compounds (semi-preparative HPLCfractions 1 to 9) were analyzed in both the negative and positive modesin order to facilitate respectively the detection of anionic (sulfated)and cationic (non-sulfated) compounds.

For fragmentation of molecules, specific ions were selected andsubmitted to MS/MS analysis using collision energy at 15V.

Mild Hydrolysis:

This method is used to remove the sulfate moiety of sulfated LCOswithout affecting the rest of the molecules (Roche et al., 1991b).Fraction A, eluting between 15 and 16 min on semi-preparative HPLC, wastransferred in a screw glass vial and dried under nitrogen flux. It wasre-dissolved two times in anhydrous methanol and dried again, in orderto remove residual water. 250 μl of 0.05M HCl in methanol was added tothe dry sample. The reaction was carried out overnight at roomtemperature. The sample was then dried again under nitrogen flux andwashed twice with anhydrous methanol, in order to remove all the acid.

Production of Milligram Quantities of Myc Factors

Purification of Myc factors from exudates of germinating spores ofGlomus intraradices and of mycorrhized roots results in extremely lowyields. Two strategies have been used to produce large amounts of thesemolecules, making use of bacterial genetic engineering.

(i) Production of Myc Factors by Rhizobium Mutants.

Rhizobia produce Nod factors that are substituted LCOs that share somestructural similarities with Myc factors. The major difference is thatMyc factors are very simple LCOs with a very limited number ofsubstitutions, essentially restricted to the possible O-sulfation of thereducing N-acetyl glucosamine residue. Our strategy was to use rhizobialmutants altered in genes coding for enzymes responsible forsubstitutions of Nod factor precursors and therefore secreting verysimple LCOs similar to Myc factors. We chose to use mutant strainsderived from rhizobial species which produce a majority of tetramericLCOs and a minority (about 10%) of pentameric LCOs, as in the case offungal Myc factors.

For the production of sulfated Myc factors we used a Sinorhizobiummeliloti nodFEnodL double mutant. The nodL mutation suppressesO-acetylation of the non-reducing GlucNAc terminal residue, and the nodFE mutation blocks the synthesis of the unsaturated 16:2 fatty acidresulting in the N-acylation with C18:1 (vaccenic) or C16:0 (palmitic)fatty acids (Ardourel et al., 1994). To increase the production of LCOs,a multicopy plasmid carrying regulatory nod genes (pMH682) wasintroduced in the mutant strain. The resulting overproducing strain, GMI6629, was grown in a liquid growth medium containing 5 μg/mltetracycline to maintain the presence of the pMH682 plasmid and luteolin(10 μM) as a nod gene inducer (Ardourel et al, 1994). When the bacterialculture reached a cell density of about 10⁹ cells per ml, Nod factorswere extracted by liquid/liquid extraction with butanol and ethylacetate (Roche et al, 1991). LCOs were then purified by HPLC on a C18reversed-phase column as previously described (Demont et al, 1993), withthe following water-acetonitrile gradient modification: a 10 minisocratic phase at 20% acetonitrile was followed by a linear gradientrunning from 20 to 65% acetonitrile for 30 min at a flow rate of 2ml/min. The peaks containing sulfated LCOs were collected between 32 and35% acetonitrile, and analyzed by mass-spectrometry. A majority of LCOswas tetrameric and a minority pentameric, as for Myc factors. LCOs wereO-sulfated at the reducing end and N-acylated with C18:1 and C16:0 fattyacids at the non-reducing end. No O-acetyl substitutions could bedetected.

For the production of non-sulfated Myc factors the strain LPR5045(pMP247) was used. It is a derivative of the R. leguminosarum bv.trifolii strain RCR5, cured from the Sym plasmid, in which a multicopyplasmid containing the common nodABCIJ genes (=pMP247) was introduced(Lugtenberg et al, 1995). This overproducing strain was grown inB-culture medium with 5 μg/ml tetracycline for maintaining the pMP247plasmid and 10 μM naringenin as a nod-gene inducer (Spaink et al, 1994).LCOs were extracted from the culture medium as described above. HPLCpurification was performed with the same C18 reversed-phase column asfor sulfated LCOs, with a 20 min isocratic phase at 26.5% acetonitrilefollowed by a linear acetonitrile-water gradient from 26.5% to 100%acetonitrile for 40 min at a flow rate of 2 ml/min. Peaks correspondingto non-sulfated LCOs were collected at about 50% acetonitrile andanalyzed by mass spectrometry. A majority of LCOs was tetrameric and aminority pentameric, as for Myc factors. N-acylation was with C18:1 andC16:0 fatty acids. No O-acetyl or O-sulfate substitutions could bedetected.

The structure of the major sulfated and non-sulfated LCOs produced bythe rhizobial mutant strains, Sinorhizobium meliloti GMI 6629 andRhizobium leguminosarum bv. trifolii LPR5045 (pMP247), respectively isrepresented below. They are perfect mimes of the Myc factors produced bythe AM fungus Glomus intraradices (see Example 2).

These Myc factors, prepared from rhizobial mutant cultures, were testedfor biological activity. Example 7 shows that a mixture of thesesulfated and non-sulfated Myc factors greatly stimulates mycorrhizaformation (FIG. 14A), demonstrating that these molecules act as genuinemycorrhizal signals.

(ii) Production of Myc Factors by the Cell Factory Approach.

These synthetic Myc factors were kindly provided by Eduardo AndresMartinez and Hugues Driguez of CERMAV CNRS laboratory in Grenoble,France. The procedure that they used was essentially as described in theliterature (Samain et al., 1999): high cell density cultivation ofrecombinant E. coli strains harboring the nodBC or nodBCH genes fromSinorhizobium meliloti afforded N^(I,II,III)-triacetyl-chitintetraoseand 6O^(I)-sulfated-N^(I,II,III)-triacetyl-chitintetraose as majorcompounds together with small amounts of their corresponding pentamers.

After extraction and purification of these compounds, selectiveN-acylation was conducted using hexadecanoic or oleic acid chlorides invarious hydro-organic solvents or by using the free acids, and theN-acylation procedure previously developed for the preparation oflipochito-oligosaccharide nodulation factors (Ohsten Rasmussen et al,2004).

The following four lipochitosaccharides were prepared:

Results

EXAMPLE 1 Purification of MYC Factors From Mycorrhized Roots Exudatesand From Germinating Spores Exudates

General Strategy

Glomus intraradices strain DAOM 197198 was used as a source of Mycfactors because this strain has a broad host-range and is used forlarge-scale industrial production of AM fungi inoculants. This strain iswell characterized and its genome is being sequenced. Two sources of Mycfactors were used in a complementary manner. Exudates from mycorrhizedroots have the advantage of permitting extraction from large volumeswith the possibility of obtaining significant amounts of Myc factors.The disadvantage of this source is that exudates contain a mixture ofcompounds from both plant and fungal origin. This is why we also usedanother source, exudates from purified germinating spores, which containonly compounds of AM fungal origin but have the disadvantage ofproducing extremely low concentrations of Myc factors.

Biologically Active Compounds Present in AM Fungal Exudates areAmphiphilic

Mycorrhized root exudates were first extracted by a liquid-liquidprocedure, with butanol and ethyl acetate. Aqueous, butanol and ethylacetate phases were checked for biological activity with MtENOD11 andVsHab bioassays: activity was found in the butanol fraction whichindicated that Myc factors are amphiphilic compounds. As shown in FIG.1, an active compound could be detected with the MtENOD11 assay by ablue staining appearing on the growing roots, and with the VsHab assayby the appearance of clear branches close to the tip of vetch roothairs.

Then the butanol extract was submitted to Solid Phase Extraction (SPE)with a reverse phase C18 column and successively eluted with 20%, 50%and 100% acetonitrile solvent. Biological activity was found in thefraction eluted by 50% acetonitrile on MtENOD11 and VsHab assaysconfirming that the active compound(s) is amphiphilic. Similar resultswere obtained with more than five independent samples of mycorrhizedroot exudates. A very slight activity on VsHab could also be observedsometimes in the 100% acetonitrile eluate, suggesting that differentcompounds could be responsible for the MtENOD11 and the VsHab responses,the compound acting on the VsHab assay being slightly more hydrophobicthan the compound acting on the MtENOD11 assay.

Exudates of germinating spores were extracted by the same liquid-liquidprocedure with butanol and ethyl acetate. The activity on both MtENOD11and VsHab assays was present only in the butanol phase. The same resultswere obtained with five independent samples of germinating spores. Wecan thus conclude that the amphiphilic compound(s) active on theMtENOD11 and VsHab assays are of AM fungal origin.

Two Types of Active Compounds in Mycorrhized Root Exudates

To further resolve the compounds active on MtENOD11 and VsHab and getinformation on their chromatographic properties, the butanol fractionhad to be analyzed by HPLC. However, the mycorrhized root exudates beinghighly contaminated by plant root compounds and by phytagel, the butanolfraction was pre-treated by SPE before the HPLC step, as described inthe preceding paragraph. The SPE fraction eluted by 50% acetonitrile,and active on the two bioassays, was then analyzed in a semi-preparativeHPLC with a reverse phase C18 column and an acetonitrile-water gradient.Fourteen fractions were collected every two minutes. A typical profileis given in FIG. 2. Each fraction was tested for activity on MtENOD11and VsHab. Fractions eluted at 30-48% of acetonitrile (ACN) (fraction A)were found to be active on MtENOD11, and fractions eluted at 64-72% ofACN (fraction B) were active on the VsHab bioassay. These data show thatit is not the same compound which is active on both bioassays. Thecompound(s) active on MtENOD11 is more hydrophilic than the one(s)active on VsHab.

It is interesting to note that the elution characteristics of thefraction active on MtENOD11 correspond well to those observed withsulfated Nod factors of Sinorhizobium meliloti and Rhizobium tropici(33-45%), which can also exhibit activity with the MtENOD11 bioassay. Onthe other hand the elution characteristics of the fraction active onVsHab correspond well to those observed with non-sulfated Nod factor ofR. leguminosarum bv. viciae (67%) and non-acetylated Nod factors ofRhizobium meliloti nodHnodL (66%) which can also exhibit activity withthe vetch bioassay. These data are compatible with the hypothesis thatthe mycorrhized root exudates contain a mixture of sulfated andnon-sulfated LCOs.

Two Types of Active Compounds in Germinating Spores Exudates

Butanol extracts from germinating spores exudates were analyzed in asemi-preparative HPLC in the same conditions as described above. As seenon FIG. 3, spore exudates also contained two types of active compounds,one more hydrophilic active on MtENOD11 (fraction A) and one morehydrophobic active on VsHal) (fraction B). The elution characteristicsof the two compounds are identical to those observed with the two activecompounds from the mycorrhized root exudates. These results indicatethat the two active compounds present in the mycorrhized root exudatesare of AM fungal origin. Their chromatographic behavior and theirbiological activities are compatible with the hypothesis that they couldcorrespond to sulfated and non-sulfated LCOs.

Modification of Activity Associated to Desulfatation of Fraction A

A mild methanolic hydrolysis of sulfated LCOs, as S. meliloti Nodfactors, has been shown to remove the sulfate group without causingother structural modifications (Roche et al., 1991b). To check whetherthe biological activity on MtENOD11 of the fraction A collected afterHPLC of butanol extracts from germinating spores exudates could be dueto a sulfated LCO, a sample of fraction A was submitted to this mildhydrolysis treatment. The treated fraction totally lost activity onMtENOD11 (see FIG. 4). Interestingly, whereas the fraction A was notoriginally active on the VsHab bioassay, the treated fraction exhibiteda clear activity on VsHab (FIG. 4). That fraction A can after mildhydrolysis gain a function, the activity on VsHab, shows that this verymild methanolic hydrolysis has not drastically degraded the activecompound of fraction A, but has simply modified it, probably by theremoval of the sulfate moiety which is a very labile O-substitution inLCOs. These data indicate that the activity of fraction A on MtENOD11could be due to sulfated LCO(s) and that the activity of the morehydrophobic fraction B on VsHab could be due to non-sulfated LCO(s).

EXAMPLE 2 Biochemical Characterization of Myc Factors

LC/MS and UPLC/MS

The different fractions obtained after the semi-preparative reversedphase HPLC of mycorrhized root extracts were individuallychromatographed on an analytical reversed phase column under ultra-highpressure (UPLC). The detection occurred through ESI-MS.

Results are shown on FIGS. 5 to 8. These figures present the ioncurrents corresponding to LCOs supposed to be present in the samples,according to the HPLC and UPLC retention times, and biological activity.If there are compounds exhibiting the requested mass within theirisotopic distribution, then they will appear on the chromatogram aspeaks. As the peaks obtained in this manner could be artefactual (peaksmight correspond to a minor compound of the isotopic profile), thecorresponding spectra are also given in the lower part of each figure.

The first eight HPLC fractions, for which chromatographic behavior andbiological activities suggested the presence of sulfated LCOs, wereanalyzed in the negative mode. According to the retention times measuredin UPLC using standard tetrameric (DP4) and pentameric (DP5) LCOs, exactmasses (error less then 10 ppm) corresponding to sulfated DP4 and DP5entities were searched in the fraction 4 which showed the highestactivity on the MtENOD11 assay. In this fraction, masses correspondingto sulfated DP4 bearing C16 acyls could easily be detected (FIG. 5). Inagreement with their respective HPLC retention times, we were able todetect in the previous fraction (fraction 3) the corresponding DP5s(FIG. 7) and in the following (fraction 5) sulfated DP4 bearing C18chains (FIG. 6). In the next fractions (6 to 8) DP3 entities have beenresearched without success. The compounds have been characterized firstusing different ion currents (fit between the called exact masses andthe expected retention times) and secondly regarding the isotopicprofile of the corresponding spectra. FIG. 8 illustrates the efficiencyof the method to detect the presence or absence of a given compound,having a specific mass. Ion current m/z 1332.6, corresponding to aputative LCO(V,C18:2,S) produced only amplification of background noise(no individual well-defined peak), whereas the ion current m/z 1334.6corresponding to a putative LCO(V,C18:1,S) clearly demonstrated thepresence of a UPLC peak containing a compound exhibiting the expectedmass (data confirmed by the recorded mass spectrum). Thus the Mycextracts contain the pentameric sulfated LCO N-acylated by C18:1 but noderivative acylated by C18:2.

By using this procedure it was not possible to detect sulfated DP4 orDP5 entities carrying O-substitutions as acetyl, carbamoyl or fucosylgroups or N-substitution as a methyl group, that are very frequentlyfound in the lipochito-oligosaccharidic Nod factors produced by diverserhizobial strains.

Fractions 7 to 11 have been analyzed then on UPLC but the detectionoccurred in the positive ESI-MS mode. The same strategy was applied: ioncall followed by analysis of the corresponding spectra.

Mycorrhized root extracts were also analyzed by LC/MS. Fractions elutingbetween 20 and 23 minutes on the semipreparative HPLC were pooled, driedunder nitrogen flux and redissolved in 150 μl of 50% ACN in water and 1%acetic acid. Solutions were directly infused into the ESI source of aQ-Tof Ultima spectrometer (Waters, US). Capillary was set at 3 kV, thecone voltage at 70V, the Rf lens at 35V. In the positive mode themolecular ions of two minor compounds at m/z 1045.5 and 1047.5 could bedetected corresponding to the sodium cationized tetrameric LCOs bearinga C16:1 or a C16:2 acyl and no O-substitution. Masses and isotopicprofiles confirmed the proposed structures.

As important amounts of contaminants (e.g. PEG) co-eluting with thesearched non-sulfated LCO compounds in fractions 9-11 prevented their MSdetection, a supplementary HPLC purification was performed. Fractions 9and 10 of the semi-preparative HPLC were pooled and injected in ananalytical C8 column and eluted using a gradient starting with 30% MeOHin water and finishing with 100% MeOH. Contaminants eluted from 1 to 15minutes. The expected LCO compounds were awaited around 20 minutes.Fractions collected between 15 and 23 minutes were separately analyzedon UPLC-MS and detection of specific ions performed based on theobserved corresponding sulfated species (DP4 and DP5; C16:0 and C18:1acyl chains). Ion call of the exact mass m/z 1027.56 (DP4,C16:1) gave ananswer in fraction eluting between 18 and 19 minutes. Retention timecompared to synthetic standards and the exact mass and isotopic profilecorresponded to the searched compound. The structure was definitivelyconfirmed by the mass spectrum recorded, that exhibited the classic Bfragmentation at m/z 400.2, 603.3, 806.4

MS/MS

Demonstrating the presence of compounds having the adequate mass at theexpected HPLC or UPLC retention time, is not sufficient to attest theirstructure. Therefore, we performed MS/MS analysis of one of the putativeLCO compound. FIG. 9 presents the comparison between the S. melilotisulfated Nod factor DP4 C16:2 in the negative mode MS/MS and the onerecorded on the “Myc factor” candidate present in the sample,LCO(IV,C16:0,S). Characteristic ions of the reducing end at m/z 503(Y₂), 605 and 706 (Y₃) are clearly detected in both case as well as thecharacteristic neutral loss of 101 amu (intracyclic rupture) startingfrom the molecular ion. The perfect fit between the two fragmentationpatterns indicates the structural affiliation of the two molecules andindicates that the sulfate group is located on the reducing glucosamineresidue whereas the fatty acyl substitution is localized on the terminalnon-reducing glucosamine residue. Since fragmentation does not producebeta-elimination ions (fatty acid ions) it is very likely that the fattyacyl substitution is an amide on the N atom of the glucosamine residue.

EXAMPLE 3 Stimulation of Lateral Root Formation by MYC Factors

Butanol extract from mycorrhized root exudates, after furtherpurification by solid phase extraction (SPE) and elution by 50%acetonitrile was incorporated into M plates and tested for growth of M.truncatula A17 seedlings. This purified Myc extract induced asignificant stimulation of lateral root formation (P=0.05). When testedon a M. truncatula dmi1 mutant (Y6) this Myc extract did not stimulatelateral root formation, indicating that this semi-purified extractcontained a Myc signal which activates lateral root formation (LRF) viathe DMI symbiotic signaling pathway (FIG. 10A).

Germinating spores exudates were extracted with ethyl acetate andbutanol. The three extracts (aqueous, ethyl acetate and butanol) werethen checked for LRF stimulation on M. truncatula A17 seedlings. Thebutanol extract stimulated LRF significantly (P=0.05) whereas theaqueous and ethyl acetate extracts were not active (FIG. 10B). Thisexperiment confirms the AM fungal origin of the amphiphilic compound(s)eliciting LRF stimulation.

The butanol extract of mycorrhized root exudates, after SPE, was furtherpurified by semi-preparative HPLC and the fractions corresponding tosulfated LCOs (fraction A, active on the MtENOD11 assay) and tonon-sulfated LCOs (fraction B, active on the VsHab assay) were collectedseparately and tested on M. truncatula A17 seedlings. The two fractionswere found to significantly stimulate LRF (FIG. 10C). These dataindicate that the Myc factors are made of a mixture of sulfated andnon-sulfated simple LCOs that are both able to stimulate the formationof lateral roots in plants.

EXAMPLE 4 Effect of MYC Factors on AM Formation in the Model Legume M.Truncatula

Synthetic Myc factors produced by the cell factory approach, asdescribed in Materials and Methods, were used to study the possibleinfluence of Myc factors on mycorrhization of roots of the model legumeMedicago truncatula by the AM fungus Glomus intraradices.

In a first series of experiments M. truncatula seedlings were grown inaxenic conditions in test tubes on a slant gellified medium poor inphosphorus and nitrogen in which Myc factors were added at aconcentration of 10⁻⁸ M. Each seedling was inoculated with 500 sterilefungal spores (Olah et al., 2005). The number of infection units (zonescontaining arbuscules, vesicules and internal hyphal networks) per plantwas counted under a binocular magnifying glass six weeks afterinoculation. Treatment by Myc factors increased the number of infectionunits per plant by 148% (see FIG. 11a ).

In a second series of experiments M. truncatula seedlings were grown ona substrate made of charred clay granulates in non-sterile conditionsand each seedling was inoculated with 50 fungal spores. Myc factors wereadded to the medium at a concentration of 10⁻⁸ M. The percentage of rootcolonization was measured by the grid intersect method three weeks afterinoculation. The percentage of mycorrhized roots in plants treated byMyc factors was 28.5% higher than in the control plants (FIG. 11b ).

Conclusions:

At low concentration (10⁻⁸ M), synthetic Myc factors stimulate AMformation on the model legume M. truncatula, providing further evidencethat the Myc factors that we have identified are genuine mycorrhizalsignals.

The fact that Myc factors efficiently stimulate AM formation in legumesopen the way to broad applications in horticulture (e.g. bean, chickpea,lentil), agriculture (e.g. soybean, pea, faba bean, alfalfa, peanut) andforestry (e.g. black locust).

EXAMPLE 5 Effect of MYC Factors on Legume Root Development

AM fungi secrete diffusible compounds which stimulate lateral rootformation (LRF) in the model legume Medicago truncatula (Olah et al.,2005). We have shown (see Example 3) that HPLC fractions containingfungal sulfated and non-sulfated LCOs elicit this LRF stimulation. Todemonstrate that this stimulation of LRF is really due to LCOs and notto fungal contaminating compounds possibly present in these HPLCfractions, we used the synthetic sulfated and non-sulfated LCOs, havingthe same structure as those detected in fungal exudates (see Example 4and Materials and Methods).

At 10⁻⁸ M pure synthetic sulfated Myc factors, or pure non-sulfatedfactors, as well as a mixture of both sulfated and non-sulfated Mycfactors, were all clearly stimulating LRF (see FIG. 12a ) showing thatboth types of compounds act as plant growth regulators. In contrast, at10⁻¹⁰ M the mixture of sulfated and non-sulfated Myc factors was stillextremely active whereas the pure compounds, sulfated or not, were notactive. These data show that a mixture of sulfated and non-sulfated Mycfactors is clearly more active than pure sulfated or non-sulfated Mycfactors.

Thus Myc factors are not only symbiotic signals activating the symbioticprogram of the host plant during early steps of mycorhization, they canalso act as genuine plant regulators, stimulate lateral root formationand influence root architecture.

From an agricultural point of view, it was important to address thequestion of the possible influence of Myc factors not only on rootbranching but also on the global development of the root system. Aftergrowing plant seedlings for 8 days on the growth medium containing ornot a mixture of sulfated and non-sulfated Myc factors, roots were cut,scanned and the root system was analyzed with the WinRhizo software.Treatment with Myc factors resulted in a 13.16% increase of total rootlength (FIG. 12b ). Treatment with Myc factors is thus able to stimulatedevelopment of the whole root system.

Conclusions:

Both sulfated and non-sulfated Myc factors are active signals, acting ata low concentration (10⁻⁸ M), but a mixture of sulfated and non-sulfatedMyc factors is clearly more active (down to 10⁻¹⁰ M).

They effectively stimulate lateral root formation and root systemdevelopment and are therefore not only symbiotic signals but also potentplant growth regulators.

These findings open the way of using these molecules in horticulture,agriculture and forestry to stimulate plant root development and plantgrowth.

EXAMPLE 6 MYC Factors Elicit Plant Responses via the DMI SymbioticSignaling Pathway

A symbiotic signaling pathway has been identified in M. truncatula withgenes coding for Nod factor perception (NFP), for calcium signaling(DMI1, DMI2 and DMI3) and a nodulation specific transcription activator(NSP1) (Catoira et al., 2000; Smit et al., 2005). Mutations in genesDMI1, DMI2 and DMI3 result in the alteration of nodule formation butalso of mycorrhiza formation, indicating that these three DMI genes areinvolved in a signalling pathway common to nodulation and mycorrhization(Catoira et al., 2000). In contrast, mutations in the NSP1 gene resultin a defect in nodulation but mycorrhization is unaffected (Catoira etal., 2000). This finding has led to the hypothesis that mycorrhizalsymbiotic signals, Myc factors, are activating the plant mycorrhizalprogramme via the DMI pathway (Catoira et al., 2000). It is impossibleto determine whether the DMI genes are involved in the stimulation of AMformation by Myc factors because dmi mutants are defective formycorrhiza formation. To address the question of the possibleinvolvement of plant symbiotic genes in the response to Myc factors wehave thus used the M. truncatula lateral root formation (LRF) assaydescribed in Examples 3 and 5.

Sulfated Myc factors exhibit some structural similarities withSinorhizobium meliloti Nod factors. To avoid possible cross talk betweenNod factor and Myc factor signaling we used non-sulfated synthetic Mycfactors. We studied the LRF stimulation response in the M. truncatulawild-type A17 line as a control, and in dmi1 (Y6), dmi2 (TR25), dmi3(TRV25) and nsp1 (B85) mutants.

As already described in Example 5, treatment of the wild-type line with10⁻⁸ M non-sulfated Myc factors resulted in a clear stimulation oflateral root formation. In contrast, in the mycorrhiza defective dmi1,dmi2 and dmi3 mutants, Myc factors did not trigger LRF stimulation (seeFIG. 13). In the nsp1 mutant which is nodulation-defective but has anormal mycorrhizal phenotype, and in which Nod factors are unable tostimulate LRF, Myc factors triggered a very clear LRF stimulation (FIG.13). These data show that Myc factors elicit plant responses downstreamof the DMI genes via a mycorrhiza specific signaling pathway, distinctfrom the Nod factor signaling pathway (NSP1).

Conclusions:

The ability of Myc factors to stimulate LRF is abolished in dmi1, dmi2and dmi3 mutants. This shows that the developmental responses induced byMyc factors are elicited via the DMI symbiotic pathway, furtherdemonstrating that Myc factors are genuine symbiotic signals.

The nodulation-specific NSP1 gene is not required for the LRFstimulation response, indicating that Myc factors trigger thisdevelopmental response via a Myc specific pathway acting through anddownstream of DMI genes and independent of the nodulation specificpathway (NSP1). This is further evidence that the Myc factors that wehave identified are genuine mycorrhizal signals.

EXAMPLE 7 Stimulation of AM Formation in Excised Transformed Roots ofCarrot, a System Used for the Production of Industrial MycorrhizalInoculants

AM fungi are obligate symbionts: they cannot propagate and form sporesin pure culture. For their growth they need to colonize roots of hostplants. This strict requirement has hampered both basic research on AMsymbiosis and the possibility of producing AM fungal inoculants on alarge scale for horticultural and agricultural purposes. An importantbreakthrough was achieved by using cultures of excised transformed rootsto grow AM fungi, making possible the production of large quantities ofsterile fungal spores (Bécard and Fortin, 1988). A system which has beenused for many years (Chabot et al., 1992) is the co-cultivation of theAM fungus Glomus intraradices strain DAOM 197198 with a carrot excisedroot clone transformed by Agrobacterium rhizogenes. This co-cultivationsystem is used namely by the Biotech company PremierTech (Québec) forthe production of commercial AM fungal inoculants. We have addressed thequestion of whether the use of Myc factors at low concentrations, as anadditive in growth media, could stimulate mycorrhization of excisedroots.

We first used a mixture of sulfated and non-sulfated Myc factors,produced by appropriate rhizobial mutants (see Materials and Methods),which was added to the growth medium at a concentration of 10⁻⁸ M.Axenic excised carrot roots were inoculated with sterile spores of G.intraradices. The percent of root length colonized by the AM fungus wasestimated by the grid line intersect method (Giovannetti and Mosse(1980). Five repetitions were used. Reading was performed with abinocular magnifying glass after eight weeks, in a double blind way.

On FIG. 14a it can be seen that the addition of a mixture of sulfatedand non-sulfated Myc factors at 10⁻⁸ M in the growth medium resulted ina very strong increase in the percent of colonization (+68.6%).

In a second experiment a mixture of sulfated and non-sulfated syntheticMyc factors was added to the growth medium at 10⁻⁸ M. Fifteenrepetitions were used. As shown on FIG. 14b after eight weeks the effectof Myc factors on the stimulation of AM formation was quite significant(+20.5%).

Conclusions:

A mixture of sulfated and non-sulfated Myc factors actively stimulatesAM formation in the roots of the non-legume carrot. This is furtherevidence that the Myc factors that we have identified and that have beensynthesized are genuine mycorrhizal signals.

Both synthetic Myc factors prepared by a biochemical procedure and Mycfactors prepared from mutant rhizobial strains are effective atstimulating mycorrhiza formation showing that these two types ofstrategies are suitable for large-scale production of Myc factors.

These data open the way to using Myc factors as additives in the growthmedia utilised for AM inoculant production by the biotech industry,using excised transformed roots.

EXAMPLE 8 Effect of MYC Factors on AM Formation in the Non-LegumeTagetes Patula

Tagetes patula, member of the Asteraceae family, has been chosen as anon-legume host plant. T. patula (French marigold) is a very populargarden plant. This species is used in companion planting for manyvegetable crops. Its root secretions are reported to kill nematodes inthe soil and it is said to repel harmful insects, such as white flyamongst tomatoes. The whole plant can be harvested when in flower anddistilled for its essential oil which is used in perfumery. T. patula isused for testing mycorrhization because it is a small plant easy tohandle and exhibiting rapid root colonization by AM fungi. The “Légiond'honneur” variety was used. Mycorrhization assays were performed bygrowing seedlings on a substrate made of particles of charred clay.Seedlings were inoculated with sterile spores of G. intraradices, andMyc factors were added at the concentration of 10⁻⁸ M.

In a first series of experiment a mixture of sulfated and non-sulfatedsynthetic Myc factors was used. The degree of mycorrhization wasestimated four weeks after inoculation by counting the number ofinfection units. Myc factor-treated plantlets had a highly significant153.5% increase of the number of infection units per plant (FIG. 15a 1).Myc factors could increase the number of infection sites either bystimulating the root system development or by increasing the density ofinfection. Indeed the treatment by Myc factors resulted in both a 49.1%increase of the root length (FIG. 15a 2), and a 30.9% increase of theinfection density (FIG. 15a 3).

In a second experiment, inoculated plants were treated either with puresulfated or non-sulfated Myc factors, or with a mixture of both. Fourweeks after inoculation the colonization rate was estimated by the gridintersect method. Results are represented in FIG. 15b . Treatment with amixture of sulfated and non-sulfated Myc factors elicited a significantdoubling of the root colonization rate (+104.5%), whereas pure sulfatedand pure non-sulfated Myc factors resulted in 42.3% and 75.4% increasesrespectively.

Conclusions:

Myc factors stimulate AM formation in a non-legume plant, furtherevidence that the Myc factors that we have identified are genuinemycorrhizal signals.

Both sulfated and non-sulfated Myc factors are active, but the mixtureof both is clearly more active.

The fact that Myc factors efficiently stimulate AM formation and rootdevelopment in a non-legume opens the way to extremely broadapplications in horticulture, agriculture and forestry.

EXAMPLE 9 Effect of MYC Factors on Seed Germination of a Non-Legume,Tomato

In previous examples, we have shown that Myc factors are not onlysymbiotic signals that activate the plant mycorrhizal program, but canalso act as plant growth regulators and stimulate root systemdevelopment at a very early stage of seedling development. We have thusinvestigated the possible influence of Myc factors on seed germinationon a non-legume plant. The tomato variety Heinz 1706 has been chosenbecause this line is well characterized and was selected for the UStomato genome sequencing project. In addition, affymetrix microarraysare available making possible gene expression profiling studies withthis tomato line.

For these studies purified synthetic Myc factors were used, eithersulfated, non-sulfated or a mixture of both. Myc factors were added togermination agar medium and poured into Petri dishes. Seeds were laid atthe surface of agar plates and incubated in the dark at 14° C., 20° C.and 28° C. The percentage of germination was scored everyday.

Experiments were performed with seeds which had been vernalized bystoring at 4° C. for at least eight weeks. The presence of Myc factorsin the germination medium, in the 10⁻⁸ M to 10⁻¹⁰ M range, resulted in avery clear stimulation of germination at 14° C. and 20° C. (see FIG.16). Each type of Myc factors, sulfated or non-sulfated, was active, butinterestingly the mixture of both types was clearly more active. Nosignificant effect of Myc factors on germination could be detected atthe highest temperature, 28° C. (data not shown). These data suggestthat this stimulation effect is operating at temperatures whichcorrespond to the range of common soil temperatures. We can hypothesizethat plants and their AM fungal symbionts have co-evolved not only forthe formation of mycorrhiza in the developing roots, but also at a veryearly stage of their interactions, germination. Both partners could havethe advantage of linking the efficiency of seed germination and earlyroot development to the presence of the fungal partner. Stimulation ofgermination was associated with a subsequent better seedling developmentas shown in FIG. 16 b.

The fact that seeds respond to Myc factors shows that plant componentsrequired for Myc factor perception (receptors) and transduction arepresent and functional in seeds. This finding opens the way to seedtreatment of crops with Myc factors in agricultural conditions. Theobservation that Myc factors are active on seed germination at extremelylow concentrations (10⁻¹⁰ M) opens the way to seed treatment technologyof a low cost (low requirement for active material) and respectingenvironment (use of extremely low concentrations of natural compounds).

Conclusions:

Both sulfated and non-sulfated Myc factors stimulate seed germination oftomato, a non-legume plant, but the mixture of both is clearly moreactive. These two types of Myc factors are therefore not only symbioticsignals but also potent plant growth regulators acting in both legumesand non-legumes.

From an agricultural point of view, these results open the way toimportant applications in horticulture, agriculture and forestry: seedtreatment by Myc factors, preferably a mixture of both sulfated andnon-sulfated ones, could improve the percentage and rate of germinationand stimulate the development of young seedlings for most cultivatedplants, the majority of which are able to establish this endomycorrhizalsymbiosis.

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We claim:
 1. A method for stimulating mycorrhization of a plant whichcomprises contacting the plant, a part, a seedling or a seed thereofwith a mixture of lipochitooligosaccharides consisting of alipochitooligosaccharide defined by the formula (I):

wherein n=2, R₁ is a saturated or monounsaturated fatty acid chaincontaining 16 or 18 carbon atoms and R₂ is H; and alipochitooligosaccharide defined by the formula (I), wherein n=3, R₁ isa saturated or monounsaturated fatty acid chain containing 16 or 18carbon atoms and R₂ is H.
 2. The method of claim 1, wherein said mixtureof lipochitooligosaccharides consists of a lipochitooligosaccharidedefined by the formula (I), wherein n is 2, R₁ is a saturated ormonounsaturated fatty acid chain containing 16 carbon atoms and R₂ is H;and a lipochitooligosaccharide defined by the formula (I), wherein n is3, R₁ is a saturated or monounsaturated fatty acid chain containing 16carbon atoms, and R₂ is H.
 3. The method of claim 1, wherein saidmixture of lipochitooligosaccharides consists of alipochitooligosaccharide defined by the formula (I), wherein n is 2, R₁is a saturated or monounsaturated fatty acid chain containing 18 carbonatoms and R₂ is H; and a lipochitooligosaccharide defined by the formula(I), wherein n is 3, R₁ is a saturated or monounsaturated fatty acidchain containing 18 carbon atoms, and R₂ is H.
 4. The method of claim 1,wherein said mixture of lipochitooligosaccharides is used at aconcentration of 10⁻⁵ to 10⁻¹² M.
 5. A method for stimulating lateralroot formation of a non-legume plant which comprises contacting saidplant, a part, a seedling or a seed thereof with a mixture oflipochitooligosaccharides consisting of a lipochitooligosaccharidedefined by the formula (I):

wherein n=2, R₁ is a saturated or monounsaturated fatty acid chaincontaining 16 or 18 carbon atoms and R₂ is H; and alipochitooligosaccharide defined by the formula (I), wherein n=3, R₁ isa saturated or monounsaturated fatty acid chain containing 16 or 18carbon atoms and R₂ is H.
 6. The method of claim 5, wherein said mixtureof lipochitooligosaccharides consists of a lipochitooligosaccharidedefined by the formula (I), wherein n is 2, R₁ is a saturated ormonounsaturated fatty acid chain containing 16 carbon atoms and R₂ is H;and a lipochitooligosaccharide defined by the formula (I), wherein n is3, R₁ is a saturated or monounsaturated fatty acid chain containing 16carbon atoms and R₂ is H.
 7. The method of claim 5, wherein said mixtureof lipochitooligosaccharides consists of a lipochitooligosaccharidedefined by the formula (I), wherein n is 2, R₁ is a saturated ormonounsaturated fatty acid chain containing 18 carbon atoms and R₂ is HSO₃H, and a lipochitooligosaccharide defined by the formula (I), whereinn is 3, R₁ is a saturated or monounsaturated fatty acid chain containing18 carbon atoms, and R₂ is H.
 8. The method of claim 5, wherein saidmixture of lipochitooligosaccharides is used at a concentration of 10⁻⁵to 10⁻¹² M.